Propagation of SARS-CoV-2 in a Closed Cell Culture Device: Potential GMP Compatible Production Platform for Live-Attenuated Vaccine Candidates under BSL-3 Conditions?

Live-attenuated SARS-CoV-2 vaccines present themselves as a promising approach for the induction of broad mucosal immunity. However, for initial safety assessment in clinical trials, virus production requires conditions meeting Good Manufacturing Practice (GMP) standards while maintaining biosafety level 3 (BSL-3) requirements. Since facilities providing the necessary complex ventilation systems to meet both requirements are rare, we here describe a possibility to reproducibly propagate SARS-CoV-2 in the automated, closed cell culture device CliniMACS Prodigy® in a common BSL-3 laboratory. In this proof-of-concept study, we observed an approximately 300-fold amplification of SARS-CoV-2 under serum-free conditions with high lot-to-lot consistency in the infectious titers obtained. With the possibility to increase production capacity to up to 3000 doses per run, this study outlines a potential fast-track approach for the production of live-attenuated vaccine candidates based on highly pathogenic viruses under GMP-like conditions that may contribute to pandemic preparedness.

Autologous regulatory T-cell transfer in refractory ulcerative colitis with concomitant primary sclerosing cholangitis.

OBJECTIVE: Ulcerative colitis (UC) is a chronic, debilitating immune-mediated disease driven by disturbed mucosal homeostasis, with an excess of intestinal effector T cells and an insufficient expansion of mucosal regulatory T cells (Tregs). We here report on the successful adoptive transfer of autologous, ex vivo expanded Tregs in a patient with refractory UC and associated primary sclerosing cholangitis (PSC), for which effective therapy is currently not available. DESIGN: The patient received a single infusion of 1×106 autologous, ex vivo expanded, polyclonal Tregs per kilogram of body weight, and the clinical, biochemical, endoscopic and histological responses were assessed 4 and 12 weeks after adoptive Treg transfer. RESULTS: The patient showed clinical, biochemical, endoscopic and histological signs of response until week 12 after adoptive Treg transfer, which was associated with an enrichment of intestinal CD3+/FoxP3+ and CD3+/IL-10+ T cells and increased mucosal transforming growth factor beta and amphiregulin levels. Moreover, there was marked improvement of PSC with reduction of liver enzymes. This pronounced effect lasted for 4 weeks before values started to increase again. CONCLUSION: These findings suggest that adoptive Treg therapy might be effective in refractory UC and might open new avenues for clinical trials in PSC. TRIAL REGISTRATION NUMBER: NCT04691232.

A One-Armed Phase I Dose Escalation Trial Design: Personalized Vaccination with IKKß-Matured, RNA-Loaded Dendritic Cells for Metastatic Uveal Melanoma.

Uveal melanoma (UM) is an orphan disease with a mortality of 80% within one year upon the development of metastatic disease. UM does hardly respond to chemotherapy and kinase inhibitors and is largely resistant to checkpoint inhibition. Hence, further therapy approaches are urgently needed. To improve clinical outcome, we designed a trial employing the 3rd generation personalized IKKß-matured RNA-transfected dendritic cell (DC) vaccine which primes T cells and in addition activates NK cells. This ongoing phase I trial [NCT04335890 (www.clinicaltrials.gov), Eudract: 2018-004390-28 (www.clinicaltrialsregister.eu)] investigates patients with treatment-naive metastatic UM. Monocytes are isolated by leukapheresis, differentiated to immature DCs, matured with a cytokine cocktail, and activated via the NF-κB pathway by electroporation with RNA encoding a constitutively active mutant of IKKß. Three types of antigen-RNA are co-electroporated: i) amplified mRNA of the tumor representing the whole transcriptome, ii) RNA encoding driver mutations identified by exome sequencing, and iii) overexpressed non-mutated tumor antigens detected by transcriptome sequencing. This highly personalized DC vaccine is applied by 9 intravenous infusions in a staggered schedule over one year. Parallel to the vaccination, standard therapy, usually an immune checkpoint blockade (ICB) as mono (anti-PD-1) or combined (anti-CTLA4 and anti-PD-1) regimen is initiated. The coordinated vaccine-induced immune response encompassing tumor-specific T cells and innate NK cells should synergize with ICB, perhaps resulting in measurable clinical responses in this resistant tumor entity. Primary outcome measures of this trial are safety, tolerability and toxicity; secondary outcome measures comprise overall survival and induction of antigen-specific T cells.

Clinical-Scale Production of CAR-T Cells for the Treatment of Melanoma Patients by mRNA Transfection of a CSPG4-Specific CAR under Full GMP Compliance.

Chimeric antigen receptor (CAR)-T cells already showed impressive clinical regressions in leukemia and lymphoma. However, the development of CAR-T cells against solid tumors lags behind. Here we present the clinical-scale production of CAR-T cells for the treatment of melanoma under full GMP compliance. In this approach a CAR, specific for chondroitin sulfate proteoglycan 4 (CSPG4) is intentionally transiently expressed by mRNA electroporation for safety reasons. The clinical-scale protocol was optimized for: (i) expansion of T cells, (ii) electroporation efficiency, (iii) viability, (iv) cryopreservation, and (v) potency. Four consistency runs resulted in CAR-T cells in clinically sufficient numbers, i.e., 2.4 × 109 CAR-expressing T cells, starting from 1.77x108 PBMCs, with an average expansion of 13.6x, an electroporation efficiency of 88.0% CAR-positive cells, a survival of 74.1% after electroporation, and a viability of 84% after cryopreservation. Purity was 98.7% CD3+ cells, with 78.1% CD3+/CD8+ T cells and with minor contaminations of 1.2% NK cells and 0.6% B cells. The resulting CAR-T cells were tested for cytolytic activity after cryopreservation and showed antigen-specific and very efficient lysis of tumor cells. Although our work is descriptive rather than investigative in nature, we expect that providing this clinically applicable protocol to generate sufficient numbers of mRNA-transfected CAR-T cells will help in moving the field of adoptive cell therapy of cancer forward.

Autophagic degradation of lamins facilitates the nuclear egress of herpes simplex virus type 1.

Dendritic cells (DCs) are crucial for the induction of potent antiviral immune responses. In contrast to immature DCs (iDCs), mature DCs (mDCs) are not permissive for infection with herpes simplex virus type 1 (HSV-1). Here, we demonstrate that HSV-1 infection of iDCs and mDCs induces autophagy, which promotes the degradation of lamin A/C, B1, and B2 in iDCs only. This in turn facilitates the nuclear egress of progeny viral capsids and thus the formation of new infectious particles. In contrast, lamin protein levels remain stable in HSV-1-infected mDCs due to an inefficient autophagic flux. Elevated protein levels of KIF1B and KIF2A in mDCs inhibited lamin degradation, likely by hampering autophagosome-lysosome fusion. Therefore, in mDCs, fewer progeny capsids were released from the nuclei into the cytosol, and fewer infectious virions were assembled. We hypothesize that inhibition of autophagic lamin degradation in mDCs represents a very powerful cellular counterstrike to inhibit the production of progeny virus and thus viral spread.

What Goes Around, Comes Around - HSV-1 Replication in Monocyte-Derived Dendritic Cells.

HSV-1 is a very successful human pathogen, known for its high sero-prevalence and the ability to infect a wide range of different cell types, including dendritic cells (DCs). As very potent antigen-presenting cells DCs play an important role in the induction of antiviral immune responses and therefore represent a strategic target for viral-mediated immune escape mechanisms. It is known that HSV-1 completes its gene expression profile in immature as well as in mature DCs, while lytic infection is only found in immature DCs (iDCs). Notably, HSV-1 infected mature DCs (mDCs) fail to release infectious progeny virions into the supernatant. Apart from HSV-1 dissemination via extracellular routes cell-to-cell spread counteracts a yet unknown mechanism by which the virus is trapped in mDCs and not released into the supernatant. The dissemination in a cell-cell contact-dependent manner enables HSV-1 to infect bystander cells without the exposure toward the extracellular environment. This supports the virus to successfully infect the host and establish latency. In this review the mechanism of HSV-1 replication in iDCs and mDCs and its immunological as well as virological implications, will be discussed.

Crystal Structure of the Extracellular Domain of the Human Dendritic Cell Surface Marker CD83.

CD83 is a type-I membrane protein and an efficient marker for identifying mature dendritic cells. Whereas membrane-bound, full-length CD83 co-stimulates the immune system, a soluble variant (sCD83), consisting of the extracellular domain only, displays strong immune-suppressive activities. Besides a prediction that sCD83 adopts a V-set Ig-like fold, however, little is known about the molecular architecture of CD83 and the mechanism by which CD83 exerts its function on dendritic cells and additional immune cells. Here, we report the crystal structure of human sCD83 up to a resolution of 1.7Å solved in three different crystal forms. Interestingly, ß-strands C', C″, and D that are typical for V-set Ig-domains could not be traced in sCD83. Mass spectrometry analyses, limited proteolysis experiments, and bioinformatics studies show that the corresponding segment displays enhanced main-chain accessibility, extraordinary low sequence conservation, and a predicted high disorder propensity. Chimeric proteins with amino acid swaps in this segment show unaltered immune-suppressive activities in a TNF-α assay when compared to wild-type sCD83. This strongly indicates that this segment does not participate in the biological activity of CD83. The crystal structure of CD83 shows the recurrent formation of dimers and trimers in the various crystal forms and reveals strong structural similarities between sCD83 and B7 family members and CD48, a signaling lymphocyte activation molecule family member. This suggests that CD83 exerts its immunological activity by mixed homotypic and heterotypic interactions as typically observed for proteins present in the immunological synapse.

The Major Immediate-Early Protein IE2 of Human Cytomegalovirus Is Sufficient to Induce Proteasomal Degradation of CD83 on Mature Dendritic Cells.

Human cytomegalovirus (HCMV) is the prototypic beta-herpesvirus and widespread throughout the human population. While infection is asymptomatic in healthy individuals, it can lead to high morbidity and mortality in immunocompromised persons. Importantly, HCMV evolved multiple strategies to interfere with immune cell function in order to establish latency in infected individuals. As mature DCs (mDCs) are antigen-presenting cells able to activate naïve T cells they play a crucial role during induction of effective antiviral immune responses. Interestingly, earlier studies demonstrated that the functionally important mDC surface molecule CD83 is down-regulated upon HCMV infection resulting in a reduced T cell stimulatory capacity of the infected cells. However, the viral effector protein and the precise mechanism of HCMV-mediated CD83 reduction remain to be discovered. Using flow cytometric analyses, we observed significant down-modulation of CD83 surface expression becoming significant already 12 h after HCMV infection. Moreover, Western bot analyses revealed that, in sharp contrast to previous studies, loss of CD83 is not restricted to the membrane-bound molecule, but also occurs intracellularly. Furthermore, inhibition of the proteasome almost completely restored CD83 surface expression during HCMV infection. Results of infection kinetics and cycloheximide-actinomycin D-chase experiments, strongly suggested that an HCMV immediate early gene product is responsible for the induction of CD83 down-modulation. Consequently, we were able to identify the major immediate early protein IE2 as the viral effector protein that induces proteasomal CD83 degradation.

Immunotherapy of Uveal Melanoma: Vaccination Against Cancer.

Uveal melanoma is the most frequently occurring primary intraocular tumor in adults, with an incidence of about 5 out of 100,000 per year, the incidence rising with increasing age (Lipski, Klin Monbl Augenheilkd 230:1005-1019, 2013; Metz et al., Klin Monbl Augenheilkd 230:686-691, 2013; Singh and Topham, Ophthalmology 110:956-961, 2003). Often diagnosed late due to a lack of early symptoms, this kind of melanoma is associated with a poor prognosis. Approximately 50 % of the patients develop distant metastases (Lipski, Klin Monbl Augenheilkd 230:1005-1019, 2013; Metz et al., Klin Monbl Augenheilkd 230:686-691, 2013; Singh and Topham, Ophthalmology 110:956-961, 2003). In sharp contrast to cutaneous melanoma, uveal melanoma shows a strong liver tropism and spreads exclusively via the hematogenous route (except for tumors with extraocular expansion) (Heindl et al., Arch Ophthalmol 128:1001-1008, 2010). The most likely reason for this observation is the lack of lymphatic vessels in the choroid and alymphatic barrier of the sclera (Schlereth et al., Exp Eye Res 125:203-209, 2014; Schroedl et al., Invest Ophthalmol Vis Sci 49:5222-5229, 2008). Due to its location in the immune-privileged eye, the uveal melanoma is widely protected from the immune system. Therefore, the goal of the approach presented here, of a "personalized vaccination therapy" is to help the immune system recognize and fight the tumor.

L Particles Transmit Viral Proteins from Herpes Simplex Virus 1-Infected Mature Dendritic Cells to Uninfected Bystander Cells, Inducing CD83 Downmodulation.

UNLABELLED: Mature dendritic cells (mDCs) are known as the most potent antigen-presenting cells (APCs) since they are also able to prime/induce naive T cells. Thus, mDCs play a pivotal role during the induction of antiviral immune responses. Remarkably, the cell surface molecule CD83, which was shown to have costimulatory properties, is targeted by herpes simplex virus 1 (HSV-1) for viral immune escape. Infection of mDCs with HSV-1 results in downmodulation of CD83, resulting in reduced T cell stimulation. In this study, we report that not only infected mDCs but also uninfected bystander cells in an infected culture show a significant CD83 reduction. We demonstrate that this effect is independent of phagocytosis and transmissible from infected to uninfected mDCs. The presence of specific viral proteins found in these uninfected bystander cells led to the hypothesis that viral proteins are transferred from infected to uninfected cells via L particles. These L particles are generated during lytic replication in parallel with full virions, called H particles. L particles contain viral proteins but lack the viral capsid and DNA. Therefore, these particles are not infectious but are able to transfer several viral proteins. Incubation of mDCs with L particles indeed reduced CD83 expression on uninfected bystander DCs, providing for the first time evidence that functional viral proteins are transmitted via L particles from infected mDCs to uninfected bystander cells, thereby inducing CD83 downmodulation. IMPORTANCE: HSV-1 has evolved a number of strategies to evade the host's immune system. Among others, HSV-1 infection of mDCs results in an inhibited T cell activation caused by degradation of CD83. Interestingly, CD83 is lost not only from HSV-1-infected mDCs but also from uninfected bystander cells. The release of so-called L particles, which contain several viral proteins but lack capsid and DNA, during infection is a common phenomenon observed among several viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus, and HSV-1. However, the detailed function of these particles is poorly understood. Here, we provide for the first time evidence that functional viral proteins can be transferred to uninfected bystander mDCs via L particles, revealing important biological functions of these particles during lytic replication. Therefore, the transfer of viral proteins by L particles to modulate uninfected bystander cells may represent an additional strategy for viral immune escape.

CD83 and GRASP55 interact in human dendritic cells.

CD83 is one of the best known surface markers for mature human dendritic cells (DCs). The full-length 45 kDa type-I membrane-bound form (mbCD83) is strongly glycosylated upon DCs maturation. As co-stimulatory properties of CD83 are attributed to mbCD83 surface expression is required for efficient T-cell stimulation by mature DCs. By yeast two-hybrid screening, we were able to identify GRASP55 as interaction partner of CD83. DCs maturation induces endogenous CD83 protein expression with simultaneous regulation of CD83 glycosylation, interaction and co-localization with GRASP55 and CD83 surface exposure. GRASP55 is especially known for its role in maintaining Golgi architecture, but also plays a role in Golgi transport of specific cargo proteins bearing a C-terminal valine residue. Here we additionally demonstrate that binding of CD83 and GRASP55 rely on the C-terminal TELV-motif of CD83. Mutation of this TELV-motif not only disrupted binding to GRASP55, but also altered the glycosylation pattern of CD83 and reduced its membrane expression. Here we show for the first time that GRASP55 interacts with CD83 shortly after induction of DC maturation and that this interaction plays a role in CD83 glycosylation as well as in surface expression of CD83 on DCs.

Herpes simplex virus type 1 ICP0 induces CD83 degradation in mature dendritic cells independent of its E3 ubiquitin ligase function.

Mature dendritic cells (mDCs) are the most potent antigen-presenting cells known today, as they are the only antigen-presenting cells able to induce naïve T-cells. Therefore, they play a crucial role during the induction of effective antiviral immune responses. Interestingly, the surface molecule CD83 expressed on mDCs is targeted by several viruses. As CD83 has been shown to exert co-stimulatory functions on mDCs, its downmodulation represents a viral immune escape mechanism. Mechanistically, it has been shown that herpes simplex virus type 1 infection leads to proteasomal degradation of CD83, resulting in a strongly diminished T-cell stimulatory capacity of the infected mDC. Previous data suggest that the viral immediate-early protein ICP0 (infected-cell protein 0) plays an important role in this process. In the present study, we showed that ICP0 is sufficient to induce CD83 degradation in the absence of any other viral factor. However, the mechanism of ICP0-mediated CD83 degradation is not yet understood. Here, we provide evidence that ubiquitination of lysine residues is, despite the published E3 ubiquitin ligase activity of ICP0, not necessary for CD83 degradation. This finding was underlined by the observation that expression of an ICP0 mutant lacking the E3 ubiquitin ligase domain in mDCs still induced CD83 degradation. Finally, inhibition of E1 activating enzyme using the specific inhibitor 4[4-(5-nitro-furan-2-ylmethylene)-3.5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester did not prevent CD83 degradation. Taken together, our data provide strong evidence that ICP0 alone induces CD83 degradation independent of its E3 ubiquitin ligase function and of the ubiquitin machinery.

Herpes simplex virus type I (HSV-1) replicates in mature dendritic cells but can only be transferred in a cell-cell contact-dependent manner.

HSV-1 is a very successful representative of the α-herpesvirus family, and ∼ 90% of the population is seropositive for this particular virus. Although the pathogen usually causes the well-known mild lesions on the lips, also, severe infections of the eye or the brain can be observed in rare cases. It is well known, that this virus can efficiently infect the most potent APCs, i.e., the DCs, in their immature and mature state. Although the infection of the iDC has been shown to be productive, infection of mMDDCs is believed to be abortive in the early phase of the viral replication cycle. In line with these findings, no virus particles can be detected in the supernatant of HSV-1-infected mMDDC. In this study, however, we show for the first time that this pathogen completes its replication cycle in mMDDCs. We detected the presence of viral gene transcripts of all three phases of the replication cycle, as well as of late viral proteins, and even the generation of small amounts of progeny virus. Although we could confirm the findings that these particles are not released into the supernatant, surprisingly, the newly generated viral particles can be passed on to Vero cells, as well as to primary keratinocytes in a cell-cell contact-dependent manner. Finally, we provide evidence that the viral gE is involved in the transfer of infectious virus from mMDDCs to other permissive cells.

Eukaryotic expression of functionally active recombinant soluble CD83 from HEK 293T cells.

The cell surface protein CD83 belongs to the immunoglobulin super family and is highly expressed on mature dendritic cells (DCs). A membrane bound and a soluble form of CD83 (sCD83) have been described. Previously, the isolation of a purified recombinant sCD83 molecule from bacterial cultures using high pressure liquid chromatography was reported. This recombinant protein reduced DC-mediated T cell proliferation in vitro and displayed an inhibitory effect in the experimental autoimmune encephalomyelitis (EAE) model. When purifying sCD83 from bacteria, however, a lipopolysaccharide fraction is frequently co-isolated with the recombinant sCD83 protein. Moreover, the subsequent separation of sCD83 from contaminating LPS is usually accompanied by a considerable loss of soluble CD83. A further disadvantage of soluble CD83 expression in prokaryotic cells is the lack of functional glycosylation. To overcome these problems, we developed an alternative strategy to express sCD83 in eukaryotic human embryonic kidney (HEK) 293 T cells. Using this system, we showed that recombinant sCD83 was LPS-free and effectively glycosylated with all three asparagine residues at least partially involved. The functionality of the expressed sCD83 protein was examined using the mixed lymphocyte reaction (MLR) assay, demonstrating a reduced DC-mediated T cell proliferation as previously reported for the sCD83 protein purified from E. coli. Thus, a new protocol for efficient eukaryotic expression and purification of sCD83 was established, which might have several advantages compared to prokaryotic expression systems.

HSV-1 upregulates the ARE-binding protein tristetraprolin in a STAT1- and p38-dependent manner in mature dendritic cells.

Dendritic cells are the sentinels of the immune system and as such represent the first-line of defense against incoming pathogens. Upon encounter with harmful antigens, these antigen-presenting cells start to mature and migrate towards the draining lymph nodes to display the antigen to T-lymphocytes, thereby eliciting the immune response of the host. Viruses, including human herpesvirus type I (HSV-1), seek to avoid such immune reactions. Therefore, they developed an arsenal of immune evasion strategies, some of which have been described earlier by our group and others. The secretion of tumor necrosis factor (TNF) represents a typical defense line of the host and it has been shown that this cytokine contributes to the inhibition of viral replication and augments the proliferation of cytotoxic T-lymphocytes. Here we report, that upon infection of mature dendritic cells, HSV-1 very strongly induces the expression of the AU-rich elements (ARE)-binding protein tristetraprolin (TTP), an mRNA-destabilizing protein. One of the best described targets of TTP is the TNF mRNA. This induction is dependent on the phosphorylation of both signal transducer and activator of transcription (STAT1) and p38 in a collaborative manner. By repressing this phosphorylation with specific inhibitors, we were able to reduce TTP mRNA levels. At the same time TNF mRNA levels were increased, suggesting that TNF mRNA is indeed a target of TTP in this setting. In summary, these data underline that HSV-1 induces TTP transcription in order to reduce TNF levels generated by infected mature dendritic cell.

Herpes simplex virus type I infection of mature dendritic cells leads to reduced LMP7-mRNA-expression levels.

Mature dendritic cells (mDCs) are the most potent antigen presenting cells within the human immune system known today. However, several viruses, including herpes simplex virus type 1 (HSV-1) have developed numerous immune escape mechanisms, such as the avoidance of peptide presentation through the major histocompatibility complex (MHC) class I to CD8(+) cytotoxic T-cells. Within the MHC class I pathway, the majority of antigenic peptides are generated by the proteasome, a multicatalytic protease complex. Upon exposure to IFN-gamma, the constitutive proteasome is partially replaced by the immunoproteasome, which contains the IFN-gamma-inducible subunits LMP2, MECL1 and LMP7. In this study, we report the downregulation of LMP7 on mRNA level in HSV-1 infected mDCs. Interestingly, this reduction was not vhs-mediated since using a virus strain lacking the vhs gene we obtained similar results. However, on protein level, LMP7-expression was not affected, which is probably due the high stability of the LMP7 protein. Also the incorporation of LMP7 into the immunoproteasome was not affected by HSV-1. However, for the in vivo situation, in which DC reside for a prolonged time period in peripheral tissues, the reduced LMP7-mRNA level could be of biological importance, since the virus could escape/hide from immune system of the host and establish latency processes.

Infection of mature dendritic cells with herpes simplex virus type 1 interferes with the interferon signaling pathway.

It has been shown that herpes simplex virus type 1 (HSV-1) blocks specific immune responses by various mechanisms. Cell lines infected with HSV-1 for instance show a severe impairment of the interferon-gamma (IFN-gamma)-induced signal transducer and activator of transcription 1 (STAT1) signaling pathway. Thus, we examined the influence of HSV-1 infection on IFN-gamma signal transduction in mature dendritic cells (mDCs). In this study, we report the down-regulation of the IFN-gamma receptor alpha chain (IFNGR1) at the mRNA level in HSV-1 infected mDCs. As a consequence, the expression of the IFNGR1 subunit on the cell surface of the infected cell was strongly reduced. Furthermore, we were able to show the inhibition of STAT1 phosphorylation following HSV-1 infection in mDCs, while protein levels of STAT1 remained constant. As a direct downstream effect of STAT1 phosphorylation, the activation of the transcription factor IRF-1 was also clearly inhibited and could no longer be induced by the addition of IFN-gamma. Additional experiments using a virus strain lacking the vhs gene suggested that the mutant virus is more sensitive to IFN-gamma as STAT1 signaling was inhibited to a lesser extent. Infection with a UV-inactivated, replication incompetent virus did not influence the STAT1 signaling pathway at all. In conclusion, we show that HSV-1 blocks IFN-gamma signaling in mDCs, which requires viral gene expression and involves the viral protein vhs.

Herpes simplex virus type 1 induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome.

Mature dendritic cells (DCs) are the most potent antigen-presenting cells within the human immune system. However, Herpes simplex virus type 1 (HSV-1) is able to interfere with DC biology and to establish latency in infected individuals. In this study, we provide new insights into the mechanism by which HSV-1 disarms DCs by the manipulation of CD83, a functionally important molecule for DC activation. Fluorescence-activated cell sorter (FACS) analyses revealed a rapid downmodulation of CD83 surface expression within 6 to 8 h after HSV-1 infection, in a manner strictly dependent on viral gene expression. Soluble CD83 enzyme-linked immunosorbent assays, together with Western blot analysis, demonstrated that CD83 rapidly disappears from the cell surface after contact with HSV-1 by a mechanism that involves protein degradation rather than shedding of CD83 from the cell surface into the medium. Infection experiments with an ICP0 deletion mutant demonstrated an important role for this viral immediate-early protein during CD83 degradation, since this particular mutant strain leads to strongly reduced CD83 degradation. This hypothesis was further strengthened by cotransfection of plasmids expressing CD83 and ICP0 into 293T cells, which led to significantly reduced accumulation of CD83. In strong contrast, transfection of plasmids expressing CD83 and a mutant ICP0 defective in its RING finger-mediated E3 ubiquitin ligase function did not reduce CD83 expression. Inhibition of the proteasome, the cellular protein degradation machinery, almost completely restored CD83 surface expression during HSV-1 infection, indicating that proteasome-mediated degradation and HSV-1 ICP0 play crucial roles in this novel viral immune escape mechanism.

Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation.

Selective gene silencing by small interfering RNAs (siRNAs) has been shown to be an efficient method for the targeted manipulation of cellular functions. Chemical transfection reagents represent the current standard technique in siRNA duplex delivery into mammalian cells. However, when trying to manipulate cells isolated from patients in clinical approaches, chemical agents might cause unwanted side effects, such as allergic reactions, or interfere with other cellular functions. In this study we describe electroporation as a suitable and efficient method for the delivery of siRNA into monocyte-derived dendritic cells (moDCs). Using a fluorescein-labeled non-silencing siRNA duplex as a model system, we carefully investigated the effects of siRNA electroporation on moDCs' viability, phenotype, migratory capacity, and ability to induce T-cell mediated immune responses. Finally, by using a standard duplex directed against the nuclear Lamins A and C we were able to demonstrate an efficient knockdown of a cellular messenger RNA in electroporated moDCs. We therefore propose siRNA electroporation into moDCs as an efficient method to manipulate DC function at large cell numbers without the use of chemical transfection reagents. This new approach represents an advantage especially in the light of clinical trials.